It has been recognized that oligonucleotides can be used to modulate mRNA expression by a mechanism that involves the complementary hybridization of relatively short oligonucleotides to mRNA such that the normal, essential functions of these intracellular nucleic acids are disrupted. Hybridization is the sequence-specific base pair hydrogen bonding of an oligonucleotide to a complementary RNA or DNA.
One deficiency of oligonucleotides for these purposes is their susceptibility to enzymatic degradation by a variety of ubiquitous nucleases which may be intracellularly and extracellularly located. Unmodified, “wild type”, oligonucleotides are not useful as therapeutic agents because they are rapidly degraded by nucleases. Therefore, modification of oligonucleotides for conferring nuclease resistance on them has been a focus of research directed towards the development of oligonucleotide therapeutics and diagnostics.
In addition to nuclease stability, the ability of an oligonucleotide to bind to a specific DNA or RNA with fidelity is a further important factor.
The relative ability of an oligonucleotide to bind to complementary nucleic acids is compared by determining the melting temperature of a particular hybridization complex. The melting temperature (Tm), a characteristic physical property of double helices, is the temperature (in ° C.) at which 50% helical versus coil (unhybridized) forms are present. Tm is measured by using UV spectroscopy to determine the formation and breakdown (melting) of hybridization. Base stacking, which occurs during hybridization, is accompanied by a reduction in UV absorption (hypochromicity). Consequently, a reduction in UV absorption indicates a higher Tm. The higher the Tm, the greater the strength of the binding of the nucleic acid strands.
Therefore, oligonucleotides modified to exhibit resistance to nucleases and to hybridize with appropriate strength and fidelity to its targeted RNA (or DNA) are greatly desired for use as research reagents, diagnostic agents and as oligonucleotide therapeutics. Various 2′-substitutions have been introduced in the sugar moiety of oligonucleotides. The nuclease resistance of these compounds has been increased by the introduction of 2′-substituents such as halo, alkoxy and allyloxy groups.
Ikehara et al. [European Journal of Biochemistry 139, 447 (1984)] have reported the synthesis of a mixed octamer containing one 2′-deoxy-2′-fluoroguanosine residue or one 2′-deoxy-2′-fluoroadenine residue. Guschlbauer and Jankowski [Nucleic Acids Res. 8, 1421 (1980)] have shown that the contribution of the 3′-endo increases with increasing electronegativity of the 2′-substituent. Thus, 2′-deoxy-2′-fluorouridine contains 85% of the C3′-endo conformer.
Furthermore, evidence has been presented which indicates that 2′-substituted-2′-deoxyadenosine polynucleotides resemble double-stranded RNA rather than DNA. Ikehara et al. [Nucleic Acids Res., 5, 3315 (1978)] have shown that a 2′-fluoro substituent in poly A, poly I, or poly C duplexed to its complement is significantly more stable than the ribonucleotide or deoxyribonucleotide poly duplex as determined by standard melting assays. Ikehara et al. [Nucleic Acids Res., 4, 4249 (1978)] have shown that a 2′-chloro or bromo substituent in poly(2′-deoxyadenylic acid) provides nuclease resistance. Eckstein et al. [Biochemistry, 11, 4336 (1972)] have reported that poly(2′-chloro-2′-deoxyuridylic acid) and poly(2′-chloro-2′-deoxycytidylic acid) are resistant to various nucleases. Inoue et al. [Nucleic Acids Research, 15, 6131 (1987)] have described the synthesis of mixed oligonucleotide sequences containing 2′-OMe substituents on every nucleotide. The mixed 2′-OMe-substituted oligonucleotide hybridized to its RNA complement as strongly as the RNA-RNA duplex which is significantly stronger than the same sequence RNA-DNA heteroduplex (Tms, 49.0 and 50.1 versus 33.0 degrees for nonamers). Shibahara et al. [Nucleic Acids Research, 17, 239 (1987)] have reported the synthesis of mixed oligonucleotides containing 2′-OMe substituents on every nucleotide. The mixed 2′-OMe-substituted oligonucleotides were designed to inhibit HIV replication.
It is believed that the composite of the hydroxyl group's steric effect, its hydrogen bonding capabilities, and its electronegativity versus the properties of the hydrogen atom is responsible for the gross structural difference between RNA and DNA. Thermal melting studies indicate that the order of duplex stability (hybridization) of 2′-methoxy oligonucleotides is in the order of RNA-RNA>RNA-DNA>DNA-DNA.
U.S. Pat. No. 5,013,830, issued May 7, 1991, discloses mixed oligonucleotides comprising an RNA portion, bearing 2′-O-alkyl substituents, conjugated to a DNA portion via a phosphodiester linkage. However, being phosphodiesters, these oligonucleotides are susceptible to nuclease cleavage.
European Patent application 339,842, filed Apr. 13, 1989, discloses 2′-O-substituted phosphorothioate oligonucleotides, including 2′-O-methylribooligonucleotide phosphorothioate derivatives. This application also discloses 2′-O-methyl phosphodiester oligonucleotides which lack nuclease resistance.
European Patent application 260,032, filed Aug. 27, 1987, discloses oligonucleotides having 2′-O-methyl substituents on the sugar moiety. This application also makes mention of other 2′-O-alkyl substituents, such as ethyl, propyl and butyl groups.
International Publication Number WO 91/06556, published May 16, 1991, discloses oligomers derivatized at the 2′ position with substituents, which are stable to nuclease activity. Specific 2′-O-substituents which were incorporated into oligonucleotides include ethoxycarbonylmethyl (ester form), and its acid, amide and substituted amide forms.
European Patent application 399,330, filed May 15, 1990, discloses nucleotides having 2′-O-alkyl substituents.
International Publication Number WO 91/15499, published Oct. 17, 1991, discloses oligonucleotides bearing 2′-O-alkyl, -alkenyl and -alkynyl substituents.
It has been recognized that nuclease resistance of oligonucleotides and fidelity of hybridization are of great importance in the development of oligonucleotide therapeutics. Oligonucleotides possessing nuclease resistance are also desired as research reagents and diagnostic agents.